Soybean selection system based on aec-resistance

ABSTRACT

A method for generating a transgenic soybean plant comprising in its genome a heterologous nucleic acid sequence of interest, comprises introducing into a soybean somatic embryo a polynucleotide encoding a functional dihydrodipicolinate synthase (DHPS) polypeptide, and a polynucleotide encoding a heterologous polypeptide of interest, operably linked to expression control sequences, wherein DHPS expressed from the introduced DHPS-encoding polynucleotide is effective to render the embryo resistant to S-2-aminoethylcysteine (2-AEC), and contacting the embryo with 2-AEC, under conditions effective for an embryo which expresses the DHPS to grow selectably and mature into a soybean plant that expresses a desired trait, and preferably includes no antibiotic resistance marker sequence.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of International Application No.PCT/US2004/020039, International filing date Jun. 23, 2004, which claimsthe benefit of U.S. Provisional Patent Application Ser. No. 60/483,103,filed on Jun. 30, 2003, the disclosures of which are incorporated hereinby reference.

FIELD OF THE INVENTION

The invention relates to methods of producing a transgenic soybean plantwith improved qualities by introducing a dihydrodipicolinate synthase(DHPS) gene conferring S-2-aminoethyl-cysteine (AEC) resistance insoybean somatic embryos as a non-antibiotic selectable marker system.The resulting improved soybean plants eliminate concerns associated withantibiotic resistance genes, and because they are AEC tolerant, they aresuitable for agricultural use with AEC as an herbicide.

BACKGROUND OF THE INVENTION

Because soybeans are a good source of oil, isoflavones and proteins,genetic engineering of soybeans for higher value cultivars hastremendous market value. Soybean transformation procedures include genegun bombardment of somatic embryos. The bombarded embryos are selectedwith a selectable marker that is part of the introduced DNA. For soybeansomatic embryo selection, the antibiotic hygromycin is often used.Market forces and health and safety concerns have created a need toeliminate such genes from improved crop varieties, but existingprocesses are time consuming. There is a need for non-antibioticselection maker genes that can eliminate these problems. Only a limitednumber of non-antibiotic resistance markers are available for plantimprovement. For example, herbicide selection systems like glyphosatemay be useful but are generally not available for use.

Soybean cellular selection systems are exceptional in that they arerecalcitrant to selection that commonly works with many plant cellularselection systems. For example, the most common cellular selectionsystem for plant systems is kanamycin resistance conferred by an nptIIgene, but after numerous failed attempts it was concluded that thisselection system cannot be accomplished with soybeans. Antibioticresistance markers have been used in seeds to produce transgenic canola,tobacco, barley, and soybean plants that express a bacterial variant ofDHPS that is insensitive to AEC. The presence of antibiotic resistancegenes in these plants is an undesirable result.

AEC is a lysine analog that naturally occurs in the mushroom Rozitescaperta (Matsumoto, 1984; Cadogan et al., 1996). It also can besynthesized using N-(tert-butoxycarbonyl)serine and ethanolamine (Arnoldet al., 1988). AEC is an inhibitor of dihydrodipicolinate synthase (DHPSor DHDPS) killing cells and tissues due to an inability to synthesizelysine (Perl et al., 1993; Ghislain et al., 1995; Vauterin et al.,2000). AEC also inhibits AK and lysine is another inhibitor of DHPS(Negrutiu et al., 1984). It is known that the DHPS enzyme of E. coli is50 fold less sensitive to AEC than plant enzymes (IC50 for lysine of 400μmM−1 mM compared to 10 μM for plants.) (Jacobs et al., 2000)

SUMMARY OF THE INVENTION

The invention provides a method for making a transgenic soybean planthaving no antibiotic resistance gene and improved soybeancharacteristics by selecting soybean somatic embryos withS-2-aminoethyl-cysteine (AEC or 2-AEC).

This invention relates, e.g., to a method for generating a transgenicsoybean plant which comprises in its genome a heterologous nucleic acidsequence of interest, comprising:

introducing into a soybean somatic embryo a polynucleotide encoding afunctional dihydrodipicolinate synthase (DHPS) polypeptide, operablylinked to a first expression control sequence, and optionally apolynucleotide encoding a heterologous polypeptide of interest, operablylinked to a second expression control sequence, wherein the first andsecond polynucleotides and expression control sequences may be the sameor different, wherein DHPS expressed from the introduced DHPS-encodingpolynucleotide is effective to render the embryo resistant toS-2-aminoethylcysteine, and

contacting the embryo produced in (a) with 2-AEC, under conditionseffective to allow an embryo which expresses the DHPS to grow selectablyand to mature into a soybean plant, while inhibiting growth ofnon-transformed embryo cells.

Progeny of a transgenic soybean plant of the invention include seeds(beans) produced by the plant, as well as variants of the original plantwhich are produced by further genetic manipulations or crosses. Forexample, a transgenic soybean plant of the invention may be heterozygousfor the DHPS or the heterologous polypeptide of interest; progeny ofthis plant include plants resulting from genetic manipulations which arehomozygous for one of both of those genes.

The invention also relates to a method for generating aherbicide-resistant transgenic soybean plant comprising: introducinginto a soybean somatic embryogenic culture a polynucleotide encoding afunctional dihydrodipicolinate synthase (DHPS) polypeptide, operablylinked to an expression control sequence, wherein DHPS expressed fromthe introduced DHPS-encoding polynucleotide is effective to render anembryo resistant to selection-effective amounts ofS-2-aminoethylcysteine (2-AEC), and to render the plant resistant toherbicide-effective amounts of AEC, and contacting the embryo withselection effective amounts of 2-AEC.

The invention also relates to a herbicide-resistant transgenic soybeanplant, or progeny thereof, comprising a polynucleotide encoding afunctional dihydrodipicolinate synthase (DHPS) polypeptide, operablylinked to an expression control sequence, wherein DHPS expressed fromthe introduced DHPS-encoding polynucleotide is effective to render thesoybean plant resistant to herbicide-effective amounts of AEC, the planthaving no antibiotic resistance marker.

The invention relates to a transgenic soybean plant comprising apolynucleotide encoding an AEC resistant DHPS that is expressible insoybean somatic embryos and a polynucleotide encoding a proteinimparting a desired trait in the soybean plant or soybean, the plantbeing free of a polynucleotide encoding a polypeptide impartingantibiotic resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrams the biosynthesis pathway of aspartate-derived aminoacids.

FIGS. 2 a 1 to 2 a-5 demonstrate that 5 mM AEC can prevent thegermination of soybean seeds; 2.5 mM is quite effective. FIGS. 2 b-1 and2 b-2 show that 500 μM AEC completely prevented the germination ofsoybean somatic embryos; 100 μM AEC was quite effective. FIG. 2 c showsthe use of 0.5 mM, 1.0, mM, 1.5, mM, 2.5 mM and 5.0 mM concentrations ofAEC and illustrates that 1.5 mM AEC is sufficient to kill proliferatingsoybean somatic embryos on ⅕th D20 medium (⅕ MS salts, ⅕ B5 vitamins and20 mg/L 2,4-D).

FIG. 2 d shows spray studies using major dicot and monocot weeds,pigweed (Amaranthus retroflexus) and giant foxtail (Setaria faberi) inaddition to tobacco. These experiments using 7 to 10 day old seedlingsshow 20 mM AEC is lethal to pigweed, foxtail and tobacco.

FIG. 3 shows a transformation vector using a Pea RbcS transit peptide totarget the E. coli DHPS to chloroplasts where the lysine biosynthesispathway is operative.

FIG. 4 is a schematic flowchart indicating that soybean somatic embryoswere induced from immature zygotic embryos on MS plates containing 40mg/L 2,4-D. Induced embryos are proliferated either on solid D20 (20mg/L 2,4-D) or FL Jack liquid medium (10 mg/L 2,4-D). Then green clumpsare bombarded with DNA coated gold particles using PD-1000 particlebombardment system (a.k.a. “Genegun”). Embryos are selected with 2.5 mMAEC on ⅕ D20 plates. Positive looking green clumps were proliferated,matured. These embryos were stained for GUS and checked for theexpression of E. coli DHPS gene to confirm the positive transformants.The resulting plants germinate and grow.

FIG. 5 shows GUS staining of DHPS transgenic embryos.

FIG. 6 demonstrates the expression of E. coli DHPS in differenttransgenic soybean somatic embryo lines and negative control embryos.

FIG. 7 shows regeneration of soybean plants via somatic embryogenesis:(a) Immature zygotic embryo cotyledon explants on medium containing 40mg/L 2,4-D (D40 medium), (b) Somatic embryo induction on D40 medium, (c)Somatic embryo proliferation on D20 medium, (d) Matured cotyledonarystage embryos on medium containing 6% maltose (MSM6), (e) Desiccation ofmatured embryos, (f) Germination on medium containing 3% sucrose (MSO3),(g) Germinated somatic embryo with well defined root and shoot system,and (h) Regenerated soybean plant transferred to soil.

FIGS. 8 a and 8 b show protocols for the transformation of soybean byparticle bombardment using liquid (8 a) and solid (8 b) media protocols.

DETAILED DESCRIPTION OF THE INVENTION

In describing preferred embodiments of the present invention, specificterminology is employed for the sake of clarity. However, the inventionis not intended to be limited to the specific terminology so selected.It is to be understood that each specific element includes all technicalequivalents, which operate in a similar manner to accomplish a similarpurpose. The embodiments of the invention may be modified or varied, andelements added or omitted, without departing from the invention, asappreciated by those skilled in the art in light of the above teachings.Each reference cited here is incorporated by reference as if each wereindividually incorporated by reference.

As used herein, the term polynucleotide is interchangeable with theterms oligonucleotide, oligomer, and nucleic acid. A polynucleotide ofthe invention may be a recombinant polynucleotide, a naturalpolynucleotide, or a synthetic or semi-synthetic polynucleotide, orcombinations thereof. Polynucleotides of the invention may be RNA, PNA,LNA, or DNA, or combinations thereof. The nucleotides of apolynucleotide of the invention can be joined via various knownlinkages, e.g., ester, sulfamate, sulfamide, phosphorothioate,phosphoramidate, methylphosphonate, carbamate, etc., depending on thedesired purpose, e.g., improved in vivo stability, etc. See, e.g., U.S.Pat. No. 5,378,825. Any desired nucleotide or nucleotide analog can beincorporated, e.g., 6-mercaptoguanine, 8-oxo-guanine, etc.

A “functional dihydrodipicolinate synthase (DHPS) polypeptide” exhibitsa detectable amount of at least one desirable function or property of aDHPS, such as those having SEQ ID NO: 3 or SEQ ID NO: 4. For example,the expression of the polypeptide renders the organism in which it isexpressed (e.g., an E. coli bacterium or a soybean somatic embryo or asoybean plant or bean) resistant to growth inhibition byS-2-aminoethylcysteine (2-AEC); and/or it is insensitive to feedbackinhibition with lysine. A skilled worker can readily determine whether agiven DHPS polypeptide exhibits a detectable amount of a desiredfunction or property, using conventional procedures.

The amino acid sequence of a DHPS polypeptide from E. coli is AAA23665[gi:145708] (SEQ ID NO: 3). 1 mftgsivaiv tpmdekgnvc raslkklidyhvasgtsaiv svgttgesat lnhdehadvv 61 mmtldladgr ipviagtgan ataeaisltqrfndsgivgc ltvtpyynrp sqeglyqhfk 121 aiaehtdlpq ilynvpsrtg cdllpetvgrlakvkniigi keatgnltrv nqikelvsdd 181 fvllsgddas aldfmqlggh gvisvttnvaardmaqmckl aaeehfaear vinqrlmplh 241 nklfvepnpi pvkwackelg lvatdtlrlpmtpitdsgre tvraalkhag ll

The amino acid sequence of a wild-type DHPS polypeptide from soybeans isAAA73555.1( gi:54832¹) (SEQ ID NO: 4). 1 mitnsaavkp nfhlpmrsfelknrtspedi kalrlitaik tpylpdgrfd leayddlvnm 61 qigqgaegvi vggttgegqlmsweehiili ahtvncfggk ikvigntgsn streaihate 121 qgfavgmhaa lhinpyygktsldgmvahfr svlsmgptii ynvpartgqd ipphviqtla 181 esvnlagvke cvgndrikqytddgivvwsg nddqchdarw gygatgvvsv asnlvpglmr 241 elmfggvnpt lnskllplidwlfhmpnpig lntalaqlgv irpvfrlpfv plpvdkrief 301 anlvkeigre hfvgnkvvevlddddfflvs ry

The amino acid sequence below (SEQ ID NO: 5) is a modified portion ofthe sequence of SEQ ID NO: 4 in which amino acid 84 is changed toarginine, starting from amino acid 54 of the soybean DHPS. Amino acid 84is changed to arginine i.e., the 30th amino acid in the sequence shown(SEQ ID NO: 5), indicated by the bold letter “R.” YDDLVNMQIG QGAEGVIVGGTTGEGQLMSR EEHIILIAHT VNCFGGKIKV IGNTGSNSTR EAIHATEQGF AVGMHAALHINPYYGKTSLD GMVAHFRSVL SMGPTIIYNV PARTGQDIPP HVIQTLAESV NLAGVKECVGNDRIKQYTDD GIVVWSGNDD QCHDARWGYG ATGVVSVASN LVPGLMRELM FGGVNPTLNSKLLPLIDWLF HMPNP

A “functional” DHPS polypeptide may comprise SEQ ID NO: 3 or SEQ ID NO:4 modified as shown in SEQ ID NO:5; or it may comprise any of a numberof variants of those sequences, either naturally occurring ordeliberately generated, provided that the changes do not substantiallyalter a function or property as discussed above.

For example, the functional DHPS polypeptide may be shorter or longerthan one of the above polypeptides. In embodiments of the invention, thevariant DHPS differs from SEQ ID NO: 3 or SEQ ID NO: 4 by one or moremodifications, which are either conservative or non-conservativemodifications (e.g., insertions, deletions, additions and/orsubstitutions). By “conservative substitutions” is meant by combinationssuch as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg;and Phe, Tyr. Variants can include, e.g., homologs, muteins andmimetics. Many types of protein modifications, includingpost-translational modifications, are included. Post-translationalmodifications include naturally occurring or synthetically produced,covalent or aggregative conjugates with other chemical moieties, e.g.,glycosyl groups, lipids, phosphates, acetyl groups, etc., as well ascleavage, such as of terminal amino acid(s). See, e.g., modificationsdisclosed in U.S. Pat. No. 5,935,835. Other functional variants maycomprise added peptide sequences, either naturally occurring orheterologous, such as, e.g., leader, signal, secretory, targeting,enzymatic etc. sequences. In one embodiment, a functional variant DHPSpolypeptide exhibits at least about 70% sequence identity (e.g., atleast about 80%, 90%, 95%, 98% or 99% sequence identity) to SEQ ID NO: 3or SEQ ID NO: 4 or SEQ ID NO: 5.

As E. Coli DHPS is naturally resistant to 2-AEC no modifications arerequired. However, with respect to soybean DHPS, as guidance as to whichamino acids can be altered without substantially altering the functionof the DHPS, the following amino acids of SEQ ID NO: 3, SEQ ID NO: 4, orSEQ ID NO: 5 have been modified to impart lysine resistance to soybeanDHPS enzyme (Silk G. W. and B. F. Matthews, 1997, Plant molecularbiology, 33:931-933) and can be expected to confer (or to contribute to)resistance to AEC (referring to SEQUENCE ID NO: 4):

amino acid 104 (ASP)

amino acid 112 (ALA).

Changes of other amino acids are less likely to affect this function ofthe protein, and thus are more amenable to alteration.

A polynucleotide which encodes a functional variant DHPS of theinvention may differ from the sequence of SEQ ID NO: 1 or SEQ ID NO: 2in a variety of ways. For example, the sequence may exhibit a percentidentity to one of those sequences of at least about 70% (e.g., at leastabout 80%, 90%, 95%, 98% or 99% sequence identity).

The comparison of sequences and determination of percent identity andsimilarity between two sequences (either polypeptide or polynucleotide)can be accomplished using any of a variety of conventional mathematicalalgorithms. (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991).

Among the many types of suitable mathematical algorithms are thosedescribed in Karlin et al. (1993) Proc. Natl. Acad. Sci. USA90:5873-5877; the algorithm incorporated into the NBLAST and XBLASTprograms (version 2.0) as described in Altschul et al. (1997) NucleicAcids Res. 25:3389-3402; the GAP program I the GCG software package(Devereux et al. (1984) Nucleic Acids Res. 12 (1):387); and thealgorithm of Myers and Miller, CABIOS (1989). Alternatively, afunctional variant polynucleotide of the invention may hybridizespecifically to a polynucleotide having SEQ ID NO: 1 or SEQ ID NO: 2under conditions of high stringency. Conditions of “high stringency,” asused herein, means, for example, hybridization in a hybridizationsolution containing, e.g., about 5×SSC, 0.5% SDS, 100 μg/ml denaturedsalmon sperm DNA and 50% formamide, at 42° C.

As used herein, the term “expression control sequence” means apolynucleotide sequence that regulates expression of a polypeptide codedfor by a polynucleotide to which it is functionally (“operably”) linked.Expression can be regulated at the level of the mRNA or polypeptide.Thus, the term expression control sequence includes mRNA-relatedelements and protein-related elements. Such elements include promoters,domains within promoters, upstream elements, enhancers, elements thatconfer tissue or cell specificity, response elements, ribosome bindingsequences, etc. An expression control sequence is operably linked to anucleotide coding sequence when the expression control sequence ispositioned in such a manner to effect or achieve expression of thecoding sequence. For example, when a promoter is operably linked 5′ to acoding sequence, expression of the coding sequence is driven by thepromoter. An expression control sequence may be linked to anotherexpression control sequence. For example, a tissue-specific expressioncontrol sequence may be linked to a basal promoter element or to anenhancer that conveys high levels of expression.

Any of a variety of expression control sequences can be used inconstructs of the invention; suitable expression control sequences willbe evident to the skilled worker.

In preferred embodiments of the invention, the “first” expressioncontrol sequence (regulating the expression of the DHPS sequence)comprises a constitutive promoter, which is expressed constitutively ina wide variety of cell types. Preferably, the promoter is sufficientlystrong to produce enough DHPS to generate sufficient levels of lysine toachieve cell growth in the presence of added AEC. Among the suitablestrong constitutive promoters/enhancers are expression control sequencesfrom ribosomal RNA promoters or, most preferably, from the CaMV 35Spromoter.

In embodiments of the invention, the “second” expression controlsequence (regulating the expression of the polypeptide of interest)comprises a tissue specific promoter, most preferably a promoter whichis active specifically in seeds (a “seed-specific” promoter). Typicalexamples are glycinin, phaseolin, conglycinin, seed lectin, napin, zeinor other seed-specific promoters

Both constitutive and tissue-specific promoters isolated from one plantfunction generally in a wide variety of other plants; thus, expressioncontrol sequences from a variety of plant species are suitable forexpression in soybeans.

In a most preferred embodiment, the “first” expression control sequence(driving expression of the DHPS sequence) comprises a constitutivepromoter; and the “second” expression control sequence (drivingexpression of the heterologous polypeptide of interest) comprises a seedspecific promoter.

In some embodiments of the invention, the DHPS-encoding sequence and/orthe sequence encoding the polypeptide of interest are not operablylinked to an expression control sequence. In such a case, thepolypeptide integrates near an endogenous expression control sequence inthe plant, and its expression is regulated by that endogenous expressioncontrol sequence. For example, an exogenously introduced polynucleotidemay integrate at a random site in the soybean genome, by illegitimaterecombination; or a DHPS sequence from E. coli may integrate at the siteof similar sequences of the endogenous soybean DHPS gene by homologousrecombination.

Additional coding sequences may be included, such as a transit peptidethat targets the polypeptide of interest to go to the desired organelle,such as a chloroplast. For example, Pea RbcS transit peptide is used totarget the E. coli DHPS to chloroplasts where the lysine biosynthesispathway is operative. Other desirable coding sequences will be apparentto a person of ordinary skill in the art.

An embryogenic culture comprising the above-discussed sequences encodingDHPS and the polypeptide of interest is “contacted” by the AEC by any ofa variety of conventional means. These include, e.g., incorporation ofAEC into the liquid or solid culture media. Methods for allowing theembryos to grow to form plants and to pollinate and produce seeds(beans) are conventional in the art.

In preferred embodiments of the invention, the DHPS and heterologousnucleic acids are stably integrated into the genome of the plant.However, in other embodiments, these nucleic acids are introduced intothe embryo on minichromosomes and are maintained stably, and aretransmitted through the germ line.

“Genome” is intended to refer to the chromosomes in the nucleus of thesoybean plant but also any autonomously replicating DNA that is passedto daughter cells, e.g. in chloroplasts and mitochondria.

Selection effective conditions are those that inhibit growth ofnon-resistant embryos, and are effective to grow transformantsselectably, e.g. a concentration and formulation of AEC sufficient toslow the growth of non-transformed embryo cells while permittingtransformed embryo cells to grow better than non-transformed embryocells, under the same conditions, preferably under conditions where thenon-transformed cells do not grow at all and transformed cells growwell, e.g. an AEC concentration of 1 mM.

In a preferred embodiment of the invention, the polynucleotide encodingthe DHPS polypeptide (which is operably linked to the first expressioncontrol sequence) and the polynucleotide encoding the polypeptide ofinterest (which is operably linked to the second expression controlsequence) are on the same plasmid. The sequence encoding the functionalDHPS polypeptide may be either upstream or downstream of the sequenceencoding the heterologous polypeptide of interest. In anotherembodiment, the two polynucleotides are on separate plasmids.

A wide variety of heterologous “polypeptides of interest” will beevident to the skilled worker. These can be, e.g., “input” genes (suchas genes conveying herbicide resistance, etc.) or “output” genes (suchas genes that provide a nutritional trait, increased oil content,proteins, etc.) Input traits are traits that benefit growers such asbetter or easier weed control, insect or disease resistance etc. Outputtraits benefit processors or consumers such as healthier or more stableoil, protein higher in limiting amino acids such as methionine, lower inoligosaccharides or phytate, etc. Among suitable polypeptides ofinterest are, e.g., omega-3 desaturase; a polypeptide providing improvedmeal amino acid compositions; a polypeptide imparting resistance to abacterium, a fungus, a virus, an insect, or a nematode; a polypeptideproviding herbicide resistance; a polypeptide affecting soybeancomposition or quality; a polypeptide that enhances nutrientutilization; or a polypeptide providing resistance to environmentalfactors (such as drought) or stress. More particularly, the polypeptidecan be phosphinothricin acetyltransferase, glyphosate resistant EPSPS,aminoglycoside phosphotransferase, hygromycin phosphotransferase,neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistantnitrilase, anthranilate synthase or glyphosatc oxidoreductase. Otherpreferred heterologous polypeptides of interest include alysophosphatidate acyl transferase (LPAT); a diacylglycerolacyltransferase (DGAT); a polypeptide which provides increased oilcontent in the soybean; delta-9 desaturase (which can result in adecreased saturated fatty acid contents in the soybean plant, resultingin palmitoleic acid accumulation in the soybean plant); and delta-12desaturase (which can result in high oleic acid content soybean oil);and DHPS.

The DHPS-encoding sequence which is operably linked to the firstexpression control sequence may take any of a variety of forms.Generally, the sequence encodes a DHPS that is resistant to AECinhibition, such as a sequence isolated from a bacterium (e.g.,Escherichia coli), or a sequence isolated from Corynebacteriumglutamicum or Nicotiana sylvestris. In a preferred embodiment, theDHPS-encoding sequence is isolated from E. coli, and is represented bySEQ ID NO: 2. In other embodiments of the invention, the DHPS-encodingsequence is a sequence that has been genetically altered from a wildtype sequence to become resistant to AEC inhibition. A variety ofpossible such alterations are discussed above. A preferred such variantis the mutant version of the endogenous soybean sequence which isdiscussed in the Examples and whose coding sequence is represented bySEQ ID NO: 1.

In embodiments of the invention, a 3′ terminator sequence (e.g., a polyAaddition and/or a RNA cleavage sequence) is located 3′ to one or both ofthe DHPS-encoding sequence and the sequence encoding the heterolgouspolypeptide of interest. The skilled worker will be aware of a number ofsuitable terminator sequences. These include, e.g., a pea RUBISCO 3′controlling sequence, a ribosomal RNA terminator, or a 3′ transcriptionregion for the nopaline synthase (NOS) gene.

In embodiments of the invention, one or both of the sequence encodingthe functional DHPS polypeptide and the sequence encoding theheterologous polypeptide of interest is operably linked to two or moreexpression control sequences. For example, a minimal promoter and somesort of enhancer would normally be needed. In embodiments of theinvention, a transgenic soybean plant produced as above is furtherbackcrossed to generate a transgenic soybean plant which is homozygousfor the sequence encoding the heterologous polypeptide of interest. Ifthe gene encoding the polypeptide of interest is dominant, it is, ofcourse, not necessary for the gene to be homozygous in order for it toexpress functional proteins. However, because soybean plants aregenerally sold as inbred lines, which breed true, it is preferable thatthe plants (and seeds derived therefrom) be homozygous for theseheterologous genes. Methods for performing suitable backcrosses areconventional, and suitable crosses will be evident to the skilledworker.

In embodiments of the invention, suitable backcrosses are performed inorder to generate a transgenic soybean plant which is homozygous for thesequence encoding the DHPS polypeptide. Homozygosity of these genes isnot required for future growth of plants (or seeds therefrom) whichexpress heterologous polypeptides of interest. In fact, the selectablemarker at this stage can even be absent. However, as discussed morefully below, in some embodiments of the invention, DHPS is expressedconstitutively, at high levels, in order to protect the plant againstinhibition by AEC, thereby allowing the use of AEC as a pesticide. Inthese embodiments, it is preferable that the DHPS is homozygous in theplant.

In embodiments of the invention, the transgenic plant is fertile.However, in other embodiments (e.g., when the leaves are to be used),the plant does not need to be fertile.

The method may include using a transformation vector, which comprisesthe DHPS coding sequence, an appropriate expression control sequence(such as a strong constitutive promoter such as the 35S promoter) andoptionally, a terminator/3′ controlling region. The vector alsocomprises a gene of interest for soybean improvement and/or may includeother genes for amplification, e.g. a gene for selection in E. coli,which can be the DHPS gene itself in an appropriate auxotroph.

The resultant soybean plant is one expressing a functional DHPS genethat is resistant to AEC levels that inhibit growth of normal soybeancells or tissues. A gene of interest may include lysophosphatidate acyltransferases (LPATs), diacylglycerol acyltransferases (DGATs), both toincrease oil contents; delta-9 desaturases to decrease saturated fattyacid contents and/or to achieve palmitoleic acid accumulation; delta-12desaturases for high oleic acid soybean oil; omega-3 desaturases; genesfor improved meal amino acid compositions; genes for disease, insect andnematode resistance; genes for herbicide tolerance; genes for droughtresistance and other genes for soybean improvement.

The DHPS gene may be a functional bacterial DHPS gene that is resistantor tolerant to AEC.

An advantage of the inventive method and plants is that selected plantsincluding AEC-resistant DHPS genes can be resistant to growth inhibitoryconcentrations of AEC in field use. This permits use of a novelherbicide that can be produced from natural sources, and may beappropriate for organic farming. See Example 4. In particular, this maybe used for weed control both in crop fields wherein the crops areselected for resistance to AEC or Lysine and Threonine or around trees,flowers and other plants, where contact to foliage is minimized as wellas around buildings and other structures.

Examples of suitable DHPS genes include: Corynebacterium glutamicum(e.g., Cremer J, Treptow C, Eggeling L, Sahm H., Regulation of enzymesof lysine biosynthesis in Corynebacterium glutamicum, J Gen Microbiol.December 1988; 134 (Pt 12):3221-9, Nucleotide sequence of the dapA genefrom Corynebacterium glutamicum; Bonnassie S, Oreglia J, Sicard A M.Nucleic Acids Res. Nov. 11, 1990; 18(21):6421), Escherichia coli (e.g.,Laber B, Gomis-Ruth F X, Romao M J, Huber R, Escherichia colidihydrodipicolinate synthase. Identification of the active site andcrystallization, Biochem J. Dec. 1, 1992; 288 (Pt 2):691-5.) orNicotiana sylvestris (e.g., Ghislain, M., Frankard, V., Jacobs, M. 1995A dinucleotide mutation in dihydrodipicolinate synthase of Nicotianasylvestris leads to lysine overproduction, Plant J., 8, 733 743).

A variety of types of expression control sequences (e.g., promoters) andterminator sequences known to one skilled in the arts that can be used,such as any strong constitutive or nearly constitutive expressioncontrol sequence and terminator. For example expression controlsequences may include a 35S promoter or ribosomal RNA gene promoters.Terminator sequences may include pea RUBISCO 3′ controlling sequences orNos terminator sequences.

A variety of AEC concentrations can be used for the selection ofbacterial DHPS transformed soybean somatic embryos, so long as thoseconcentrations are in an amount that would inhibit the growth of soybeanseeds or somatic embryos without the AEC resistant DHPS gene. Forexample, at least about 0.1 mM or higher may be sufficient to inhibitgrowth, and 1 mM or higher can stop all cell growth. Higherconcentrations than about 10 mM may inhibit growth even in transformedembryos. AEC concentrations for selection can be from about 0.5 mM toabout 5 mM, 1.0 mM to about 5 mM, 2.5 mM to about 5 mM, or more than orless than about 0.5 mM, 1 mM, 1.5, mM, 2 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0mM, 4.0 mM or 5.0 mM. Concentrations in the range up to about 10 mM or20 mM may be suitable for selection in some circumstances. In latergrowth stages and including field use, the higher concentrations of AECmay be preferred, in conjunction with surfactants or other uptakeenhancers.

A variety of transformation protocols may be used with the inventiveprocess. For example, FIGS. 8 a and 8 b show the transformation ofsoybean by particle bombardment using liquid and solid media protocolsusing somatic embryos (Trick et al., 1997). Once embryogenic culturesare established, one can get plenty of material with a liquid mediumbased protocol for transformation. However, with older cultures theregenerated plants are mostly sterile (Hazel et al., 1998). With solidmedium-based protocols somatic embryos need to induced regularly. Thematuration of somatic embryos on MSM6 medium takes much longer than withliquid medium based protocols with FL-Superlite (Samoylov et al., 1998).

Targeting cells that can give rise to whole plants for transformationand selection of whole plants is quite difficult with soybeans.Kanamycin selection which works well with most plants does not work atall with soybeans. The precise reasons of why some plants such assoybeans are so recalcitrant to standard transformation and regenerationsystems are unknown, except that it is thought that many more cells inplants such as soybeans are terminally differentiated compared witheasier plants such as potatoes or tobacco.

Suitable transformation vectors known to one skilled in the art can beused with the inventive process. For example, the Binary vectorpCAMBIA-1201, Genbank # AF234293, which may be obtained from Cambiagroup in Australia. Before cloning pCambia1201, the DHPS gene can becloned along with pea rbcs chloroplast transit peptide into anotherbinary vector pKalyx. This is done to clone the E. coli DHPS gene alongwith transit peptide under 35S promoter. This vector has a Nosterminator.

The accession number is gi|145707|gb|M12844.1 for the following E. coliDHPS sequence ( SEQ ID NO: 2): 1 ccaggcgact gtcttcaata ttacagccgcaactactgac atgacgggtg atggtgttca 61 caattccacg gcgatcggca cccaacgcagtgatcaccag ataatgtgtt gcgatgacag 121 tgtcaaactg gttattcctt taaggggtgagttgttctta aggaaagcat aaaaaaaaca 181 tgcatacaac aatcagaacg gttctgtctgcttgctttta atgccatacc aaacgtacca 241 ttgagacact tgtttgcaca gaggatggcccatgttcacg ggaagtattg tcgcgattgt 301 tactccgatg gatgaaaaag gtaatgtctgtcgggctagc ttgaaaaaac tgattgatta 361 tcatgtcgcc agcggtactt cggcgatcgtttctgttggc accactggcg agtccgctac 421 cttaaatcat gacgaacatg ctgatgtggtgatgatgacg ctggatctgg ctgatgggcg 481 cattccggta attgccggga ccggcgctaacgctactgcg gaagccatta gcctgacgca 541 gcgcttcaat gacagtggta tcgtcggctgcctgacggta accccttact acaatcgtcc 601 gtcgcaagaa ggtttgtatc agcatttcaaagccatcgct gagcatactg acctgccgca 661 aattctgtat aatgtgccgt cccgtactggctgcgatctg ctcccggaaa cggtgggccg 721 tctggcgaaa gtaaaaaata ttatcggaatcaaagaggca acagggaact taacgcgtgt 781 aaaccagatc aaagagctgg tttcagatgattttgttctg ctgagcggcg atgatgcgag 841 cgcgctggac ttcatgcaat tgggcggtcatggggttatt tccgttacga ctaacgtcgc 901 agcgcgtgat atggcccaga tgtgcaaactggcagcagaa gaacattttg ccgaggcacg 961 cgttattaat cagcgtctga tgccattacacaacaaacta tttgtcgaac ccaatccaat 1021 cccggtgaaa tgggcatgta aggaactgggtcttgtggcg accgatacgc tgcgcctgcc 1081 aatgacacca atcaccgaca gtggtcgtgagacggtcaga gcggcgctta agcatgccgg 1141 tttgctgtaa agtttaggga gatttgatggcttactctgt tcaaaagtcg cgcctgg

The coding sequence which starts at 272 and ends at 1150 was used.

The effect of AEC on proliferating somatic embryos were conducted inboth liquid and solid media and methylated seed oil may be added as asurfactant to facilitate AEC absorption. MSO concentration may be about0.0007%. However, even without MSO, AEC may be effective in growthinhibition of soybean somatic embryos.

There are a variety of different sources/suppliers from which to obtainAEC, e.g., Sigma, and there are several published procedures for itssynthesis. AEC is a lysine analog that naturally occurs in the mushroomRozites caperta (Matsumoto, 1984; Cadogan et al., 1996) (also, seeExample 4).

Suitable transformation methods known to one skilled in the art can beused. One of the well established soybean transformation procedures isgene gun bombardment of somatic embryos (Finer and McMullen, 1991). Thebombarded embryos are be selected with a selectable marker on theintroduced DNA. So far for soybean selection the antibiotic hygromycinis the one mostly in use. Known methods are adapted to eliminateantibiotic resistance genes commonly included on existing vectors, whilepermitting amplification (growth) of the vector plasmids. For example,gene gun transformation has traditionally used the entire cloningplasmid including an antibiotic resistance gene for selection in E. colibut a restriction site can be included so that after amplification, theantibiotic resistance gene section can be cut out, leaving the DHPS geneand the gene of interest to be used for bombardment into the cells.

For another example, with Agrobacterium mediated transformation normallyonly DNA between the T-left and T-right borders is incorporated intoplant chromosomes. Accordingly, the antibiotic resistance gene(s) forbacterial selection can be placed outside these borders so that they arenot inserted into the target cells by the transformation process.

For a third example, one can flank genes that are not wanted inpost-transformation target plant chromosomes with CRE sites, so thatcrossing the resulting transgenic plants containing inserted LOX geneswill cause the genes flanked with CRE sites to be cleaved out insubsequent generations.

No antibiotic resistance genes are needed for cloning in E. coliauxotrophic for DHPS. A preferred method is to amplify suchantibiotic-resistance gene-free vectors in E. coli, and then use genegun bombardment for transformation.

An improved soybean seed according to the invention can be planted in anopen field. AEC is applied during germination, growth, and/or maturationto eliminate weeds. Soybeans are harvested from the mature plant, havingimproved output traits. The DHPS gene used to confer AEC resistance canbe derived from a soybean, and the AEC that is used can be obtained fromnatural sources, permitting the harvested soybeans to be marketed morereadily than transgenic soybeans having heterologous genes, antibioticresistance genes, or glyphosate or other herbicide resistance genes thatrequire use of synthetic herbicides.

EXAMPLES Example 1

As a first step in the selection, different concentrations of AEC wereused to see if it can kill the proliferating soybean somatic embryos aswell as tobacco leaves and germinating tobacco and soybean seeds andgerminating soybean somatic embryos. The effect of AEC is clearly seenin all these cases with varying concentrations. 100 μm AEC is sufficientto completely inhibit the germination of soybean somatic embryos whereasfor germinating soybean seeds and tobacco seeds 2.5 mM AEC is required.For proliferating soybean somatic embryo concentrations up to 1 mM AECis needed along with 0.0075% methylated seed oil to kill them. Tofurther confirm that AEC is acting on the above mentioned tissues, theE. coli DHDPS gene was cloned and put into the binary vector p1201 withthe pea RBCs transit peptide under the CaMV 35S promoter.

Proliferating soybean somatic embryos were shot with a gene gun usingthe E. coli DHDPS gene construct and the embryos were selected on bothsolid D20 and liquid F1 cv Jack proliferating media with 1 mM to 2.5 mMAEC with 0.0075% methylated seed oil. The positive looking green clumpswere put on the MSM6AC maturation medium. Some of the embryos have beenshown to contain an introduced GUS gene indicating the soybeantransformation with AEC as a selective agent can be achieved.

Example 2

Lysine-Threonine and Lysine-Analog S-(2-aminoethyl)-L-cysteine asAlternative Non-Antibiotic Selection System for Soybean Somatic Embryos.

Introduction

Plant cells and tissues are normally killed by exposure toS-2-aninoethyl-cysteine (AEC), which prevents lysine synthesis throughinhibition of the plant enzyme dihydrodipicolinate synthase (DHPS). E.Coli DHPS is not inhibited by AEC. A vector for producing transgenicsoybean plants encodes E. Coli DHPS. Soybean cells and tissues that aresuccessfully transfected with the vector can be identified by exposureto AEC, which kills cells and tissues not containing the gene encodingE. Coli DHPS.

Procedure

An AEC selection system for soybean seeds with precise genetic changesand model system seeds (for rapid system validation) were germinated on2 mM and 5 mM AEC containing agar plates. On the control plates withoutAEC all the seeds germinated, however only 10% of the soybean seedsgerminated on 2 mM AEC plates and none on 5 mM plates. Not a singlemodel system seed germinated at either of these concentrations.

The effect of AEC on soybean somatic embryos were checked at two stages(1) during the germination (2) during proliferation. The AEC effectduring germination was very effective with concentrations above 100 μMwere enough to prevent the germination completely.

However, selection during proliferation is always a better way asselected clones can be proliferated and the untransformed embryos can bekilled in the earlier stages itself. Unfortunately concentrations up to20 mM were not sufficient to kill most of the proliferating embryos,though some of the embryos turned yellowish and developed brown spots,but many of them continued to proliferate like controls even after twochanges of the medium, once every 15 days. To find out if surfactantscan help in AEC absorption during proliferation a methylated seed oilbased surfactant was used. A series of experiments were run with liquidproliferation medium to determine a suitable surfactant concentrationsas the 0.2% which was used initially killed the cells even without AECaddition to the medium. It was determined that a 0.005% concentration issuitable to use with AEC. However on solid medium with 0.2% surfactantboth controls and 10 mM AEC cultures remained green with growth for twoweeks before they died. The cultures on plates with 20 mM AEC died in 4days indicating solid medium selection can be useful.

To test if the modified soy DHDPS can become insensitive to AEC andwhether 18:1 desaturases can function as a 16:1 desaturase, experimentswere conduced with E. Coli auxotrophs. A DHDPS auxotroph was obtainedfrom the E. coli genetic center that can be rescued on medium withoutdiaminopimelic acid when transformed with lysine insensitive DHPS andnot with the wild type gene. Similarly, the E. coli auxotroph (fabA/fadRmutant) was obtained which can be complimented by C₁₄ and C₁₆ ACPdesaturases but not by stearoyl-ACP desaturases. The primers requiredfor cloning were designated and obtained from soybean genes. A suitableE. coli expression vector was obtained to express these genes in therespective auxotrophs.

Summary

Experiments were conducted with different tissues of soybean todetermine whether LT and AEC can be used as selection agents. Both AECand LT can inhibit seed as well as soybean somatic embryos (SSE)germination. AEC concentrations of 1.5 to 2.5 mM are effective forselection in presence of 0.075% MSO (methylated seed oil) as surfactant.However LT (up to 10 mM) were not able to inhibit growth ofproliferating SSE. Soybean SE and tobacco transformed with wild-type E.coli DHPS are found to be resistant to normally lethal concentrations ofAEC. Green proliferating transgenic somatic embryos were found to beselected at the concentration level of 2.5 mM AEC. Similarly, selectionat low micro molar AEC and 1.5 mM LT resulted in transgenic tobaccoshoots. These transgenic SS embryos and tobacco shoots were alsoobserved to be GUS positive indicating resistance to AEC can be used asa selection system for soybean and tobacco.

Results

To determine whether AEC can be developed as a selectable marker forsoybeans the effect of different concentrations of AEC was examined onthe germination of soybean seeds and matured soybean somatic embryos andon proliferating soybean somatic embryos. The effect of AEC is clearlyseen in all these cases with varying concentrations. The effect of AECwas also examined as a herbicide against pigweed, foxtail and tobacco.

FIGS. 2 a 1 to 2 a-5 demonstrate that 5 mM AEC can prevent thegermination of soybean seeds; 2.5 mM is quite effective. FIGS. 2 b-1 and2 b-2 show that 500 μM AEC completely prevented the germination ofsoybean somatic embryos; 100 μM AEC was quite effective. FIG. 2 c showsthe use of 0.5 mM, 1.0, mM, 1.5, mM, 2.5 mM and 5.0 mM concentrations ofAEC and illustrates that 1.5 mM AEC is sufficient to kill proliferatingsoybean somatic embryos on ⅕th D20 medium (⅕ MS salts, ⅕ B5 vitamins and20 mg/L 2,4-D). FIG. 2 d shows spray studies using major dicot andmonocot weeds, pigweed (Amaranthus retroflexus) and giant foxtail(Setaria faberi) in addition to tobacco. These experiments using 7 to 10day old seedlings show 20 mM AEC is lethal to pigweed, foxtail andtobacco.

FIG. 3 shows a transformation vector using Pea RbcS transit peptide totarget the E. coli DHPS to chloroplasts where the lysine biosynthesispathway is operative.

In FIG. 4, SS embryos were induced from immature zygotic embryos on MSplates containing 40 mg/L 2,4-D. Induced embryos are proliferated eitheron solid D20 (20 mg/L 2,4-D) or FL Jack liquid medium (10 mg/L 2,4-D).Then green clumps are bombarded with DNA coated gold particles usingPD-1000 particle bombardment system (a.k.a. “Genegun”). Embryos areselected with 2.5 mM AEC on ⅕ D20 plates. Positive looking green clumpswere proliferated, matured. These embryos were stained for GUS andchecked for the expression of E. coli DHPS gene to confirm the positivetransformants.

FIG. 5 shows GUS staining of DHPS transgenic embryos.

FIG. 6 demonstrates the expression of E. coli DHPS in differenttransgenic soybean somatic embryo lines and negative control embryos.

Discussion

The AEC selection results showed that AEC can be successfully used as aselection marker for soybeans. The added advantage of this selectionsystem besides being a non-antibiotic selection marker is that it couldincrease the levels of essential amino acid lysine. No problems werefaced in the maturation and germination of these transgenic embryos.When the effect of AEC was tested at the whole plant level, 20 mM AECwas sufficient to kill fox tail and tobacco and effectively preventedthe growth of pig weed.

Conclusion

The lysine analog AEC is a successful non-antibiotic selectable markerfor the transformation of soybean somatic embryos.

Example 3

SEQ ID No. 1: ctttggactctgaagctatgatgacttggtgaatatgcagattggacaaggggctgaaggtgttattgttggtgggacaactggtgaaggccaattaatgagccgggaagagcacataatacttattgctcatacagtcaactgttttggtgggaaaattaaggttattggaaatactggaagcaactccaccagggaagcaattcatgccactgagcagggttttgctgttggaatgcatgctgcccttcacataaacccttactatggcaaaacctccttggatggtatggttgctcactttcgaagtgtgctttccatgggacccacaataatctacaatgtgcctgcacggaccggacaagacattcctccgcatgtaattcaaaccttagctgaaagtgttaacctggctggtgtcaaggagtgtgtgggaaatgaccgaatcaaacagtatacagatgatggaattgttgtgtggagtgggaatgatgatcaatgtcatgatgctagatggggttatggggctaccggagtggtatctgttgcgagcaacctggttcccggtttaatgcgagaactcatgtttggcggtgtaaaccctactctaaattctaaactcttgcctctgattgactggcttttccacatgccaaaccccatnggtttgaacactgctcttgctcaacttggggncatc

The above sequence was derived from the wild-type soybean sequenceencoding the polypeptide of SEQ ID NO:4. The nucleotide sequence shownas SEQ ID NO:1 corresponds to sequences encoding the polypeptide of SEQID NO:5, a genetically altered AEC resistant DHPS sequence derived fromsoybean. The soybean DHPS sequence shown as SEQ ID NO:1 was modifiedfrom the wild-type to comprise a change of tgg to cgg at the indicatedposition in bold type. According to the invention, a soybean DHPS codingsequence comprising the indicated modification provides the desired AECresistance.

The sequence was prepared using techniques well know to one skilled inthe art. (e.g., Vauterin M, Frankard V, Jacobs M., Functional rescue ofa bacterial dapA auxotroph with a plant cDNA library selects for mutantclones encoding a feedback-insensitive dihydrodipicolinate synthase,Plant J. February 2000; 21(3):239-48.; and Shaver, J., Bittel, D.,Sellner, J., Frisch, D., Somers, D., Gengenbach, B. 1996 Single-aminoacid substitutions eliminate lysine inhibition of maizedihydrodipicolinate synthase. Proc. Natl Acad. Sci. USA , 93, 1962 66).

It was shown in Arabidopsis, and populous deltoidsXpopulus tricho carpamodification of tryptophan53 (W) to Arginine53 (R) made the DHPS enzymefrom these two plants insensitive to lysine concentrations up to 1 mM.E. coli auxotroph AT997 was rescued using these modified genes. Thistryptophan residue is present in the consensus sequence MSWDEHI (SEQ IDNO: 6) in several plant DHPS genes (Vauterin et al.,2000). Clonesexpressing the insensitive plant DHPS enzymes were resistant to 2.5 mM2-AEC. With our modified soybean DHPS gene we obtained similar results.We also targeted the tryptophan residue present in the consensussequence MSWDEHI (SEQ ID NO: 6) in the soybean DHPS gene to convert itto arginine. With the modified gene we rescued the E. coli auxotrophAT998 and found that the rescued auxotroph grows well on minimal mediaplates with 0.5 mM 2-AEC concentrations. Native soybean DHPS gene failedto rescue the auxotroph while the modified soybean DHPS gene and nativeE. coli DHPS were successful in rescuing the mutant (data not shown).

Example 4

Method for Weed Control

The purpose of this example is to illustrate a method for weed controlthat has several potential advantages over existing methods.

Background:

The broadest class of major herbicides is the acetolactate synthase(ALS) inhibitors including the sulfonylureas, imidazolinones,sulfonamides and pyrimidinylthiobenzoate. DuPont and Syngenta havecommercialized no less than 12 different sulfonylureas that target ALS.American Cyanamid (now BASF) have at least 6 different imidazolinonesherbicides on the market all of which also target ALS (Shaner andO'Connor, 1991). ALS catalyzes the first step in the biosynthesis of theessential amino acids valine, isoleucine and leucine, as set forth inFIG. 9.

Via a mutagenesis program, DuPont has been able to produce an alteredALS in soybeans that is resistant to sulfonylureas and from this havedeveloped sulfonylurea tolerant soybeans (STS) (Sebastian, 1990).

Roundup (glyphosate) also functions as an amino acid biosynthesisinhibitor targeting 5-enoylpyruvylshikimate 3-phosphate (EPSP) blockingaromatic amino acid biosynthesis, as shown in FIG. 10.

All of these herbicides kill plants by starving them of essential aminoacids but do not affect animals as essential amino acids in animals areobtained via their diets.

The selective agent AEC, the chemical structure of which is shown inFIG. 11, can be synthesized using N-(tert-butoxycarbonyl)serine andethanolamine (Arnold et al., 1988). AEC is an inhibitor ofdihydrodipicolinate synthase (DHPS) killing cells and tissues due to aninability to synthesize lysine (Perl et al., 1993; Ghislain et al.,1995; Vauterin et al., 2000). AEC also inhibits AK and lysine, which isalso an inhibitor of DHPS (Negrutiu et al., 1984). AEC is shown below tobe a more effective more effect herbicidal agent than lysine orlysine+threonine.

Results:

A major dicot and monocot weed of tobacco, pigweed (Amaranthusretroflexus) and giant foxtail (Setaria faberi) was used in addition toa model crop plant, tobacco. Initial experiments using 7 to 10 day oldseedlings showed 10 mM lysine and threonine and above is lethal topigweed, foxtail and tobacco. For larger plants grown in the green house20 mM lysine and threonine or 40 mM lysine alone are needed foreffective weed control. To increase uptake of these compounds, up to0.2% Silwett (surfactant) was used. Silwett at 0.5% and above kills theplants. Silwett was not found to be a very effective surfactant. Studieswith cultured cells and seedlings indicate that 100 μM levels of AEC areeffective for selection.

LITERATURE CITED

Each reference cited here is incorporated by reference as if each wereindividually incorporated by reference.

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1. A method for generating a transgenic soybean plant which comprises inits genome a heterologous nucleic acid sequence of interest, comprising:(a) introducing into a soybean somatic embryo a polynucleotide encodinga functional dihydrodipicolinate synthase (DHPS) polypeptide, operablylinked to a first expression control sequence, wherein DHPS expressedfrom the introduced DHPS-encoding polynucleotide is effective to renderthe embryo resistant to S-2-aminoethylcysteine (2-AEC), and (b)contacting the embryo with 2-AEC under conditions inhibiting growth of anon-resistant embryo but permitting an embryo which expresses the DHPSto grow selectably and mature into a soybean plant.
 2. The method ofclaim 1, further comprising introducing a polynucleotide encoding apolypeptide of interest, operably linked to a second expression controlsequence wherein the first and second polynucleotides and theirexpression control sequences may be the same or different.
 3. The methodof claim 1, wherein the DHPS-encoding polynucleotide and/or thepolypeptide of interest-encoding polynucleotide are stably integratedinto the genome.
 4. The method of claim 1, wherein the polynucleotideencoding the DHPS polypeptide and the polynucleotide encoding thepolypeptide of interest are on the same molecule.
 5. The method of claim1, wherein the polynucleotide encoding the DHPS polypeptide, operablylinked to the first expression control sequence, and the polynucleotideencoding the polypeptide of interest, operably linked to the secondexpression control sequence, are on separate molecules.
 6. The method ofclaim 1, wherein the first expression control sequence is a constitutivepromoter.
 7. The method of claim 6, wherein the first expression controlsequence comprises a cauliflower mosaic virus CaMV 35S promoter or aribosomal RNA promoter.
 8. The method of claim 2, wherein the secondexpression control sequence is a seed-specific promoter.
 9. The methodof claim 8, wherein the second expression control sequence comprises aglycinin, phaseolin, conglycinin, seed lectin, napin, zein or otherseed-specific promoter.
 10. The method of claim 2, wherein the sequenceencoding the functional DHPS polypeptide is upstream of the sequenceencoding the polypeptide of interest.
 11. The method of claim 2, whereinthe sequence encoding the functional DHPS polypeptide is downstream ofthe sequence encoding the polypeptide of interest.
 12. The method ofclaim 2, wherein the heterologous polypeptide of interest is omega-3desaturase; a polypeptide for improved amino acid compositions; apolypeptide imparting resistance to a bacterium, a fungus, a virus, aninsect, or a nematode; a herbicide resistance polypeptide; a polypeptideaffecting soybean composition or quality; a nutrient utilizationpolypeptide; an environmental or stress resistance polypeptide; and/or adrought resistance polypeptide.
 13. The method of claim 2, wherein thepolypeptide is phosphinothricin acetyltransferase, glyphosate resistantEPSPS, aminoglycoside phosphotransferase, dalapon dehalogenase,bromoxynil resistant nitrilase, anthranilate synthase and glyphosateoxidoreductase.
 14. The method of claim 2, wherein the polypeptide ofinterest is a lysophosphatidate acyl transferase (LPAT).
 15. The methodof claim 2, wherein the polypeptide of interest is a diacylglycerolacyltransferase (DGAT).
 16. The method of claim 2, wherein thepolypeptide of interest provides increased oil content in the soybean.17. The method of claim 2, wherein the polypeptide of interest isdelta-9 desaturase.
 18. The method of claim 17, wherein expression ofthe delta-9 desaturase activity results in a decreased saturated fattyacid contents in the soybean plant.
 19. The method of claim 18, whereinthe decreased fatty acid content results in palmitoleic acidaccumulation in the soybean plant.
 20. The method of claim 2, whereinthe polypeptide of interest is delta-12 desaturase.
 21. The method ofclaim 20, wherein expression of the delta-12 desaturase results in higholeic acid content soybean oil.
 22. The method of claim 21, wherein thepolypeptide of interest is a functional DHPS expressible in soybeanplant and seed.
 23. The method of claim 22, wherein the polypeptide ofinterest is the same as the DHPS-encoding sequence.
 24. The method ofclaim 1, wherein the DHPS-encoding sequence encodes a bacterial DHPSthat is resistant to AEC inhibition.
 25. The method of claim 1, whereinthe DHPS-encoding sequence is isolated from an organism selected fromthe group consisting of Corynebacterium glutamicum, Escherichia coli andNicotiana sylvestris.
 26. The method of claim 25, wherein theDHPS-encoding compirses the coding sequence represented by SEQ ID NO: 2.27. The method of claim 1, wherein the DHPS-encoding sequence has beengenetically altered to become resistant to AEC inhibition.
 28. Themethod of claim 1, wherein the DHPS-encoding sequence is isolated fromsoybean and has been genetically altered to be resistant to AECinhibition.
 29. The method of claim 28, wherein the DHPS-encodingsequence comprises the sequence represented by SEQ ID NO:
 1. 30. Themethod of claim 1, wherein a 3′ terminator sequence is located 3′ to theDHPS-encoding sequence.
 31. The method of claim 30, wherein the 3′terminator sequence is a pea RUBISCO 3′ controlling sequence, aribosomal RNA terminator, or a 3′ transcription region for the nopalinesynthase (NOS) gene.
 32. The method of claim 2, wherein one or both ofthe sequence encoding the functional DHPS polypeptide and the sequenceencoding the heterologous polypeptide of interest is operably linked totwo or more expression control sequences.
 33. The method of claim 2,wherein the transgenic soybean plant is backcrossed so as to generate atransgenic soybean plant which is homozygous for the sequence encodingthe heterologous polypeptide of interest.
 34. The method of claim 1,further comprising backcrossing the transgenic soybean plant to generatea transgenic soybean plant which is homozygous for the sequence encodingthe DHPS polypeptide.
 35. The method of claim 1, wherein the transgenicplant is fertile.
 36. The method of claim 1, further comprisingpropagating in a bacterium a plasmid comprising the DHPS-encodingsequence, the expression control sequence, and a polynucleotide encodinga selectable or screenable marker for bacterial culture, operably linkedto an expression control sequence.
 37. The method of claim 36, whereinthe marker for bacterial culture is an nptII gene, a bla gene, a nptIgene, a dhfr gene, an aphIV gene, an aacC3 gene, an aacC4 gene or a GUSgene.
 38. The method of claim 36, wherein the polynucleotide encoding amarker for bacterial culture is the DHPS encoding sequence and theculture is an AEC-sensitive E. coli auxotroph.
 39. The method of claim36 wherein the polynucleotide encoding a marker for bacterial cultureimparts antibiotic resistance, and further comprising cleaving theantibiotic resistance polynucleotide from the plasmid prior tointroducing the plasmid sequences into the somatic soybean embryos. 40.The method of claim 39, further comprising cleaving the antibioticresistance polypeptide from the DHPS-encoding sequence portion of theplasmid by action of a restriction enzyme.
 41. The method of claim 1,wherein the embryo is contacted with a concentration of AEC from about0.1 to about 20 mM.
 42. The method of claim 1, wherein the embryo iscontacted with a concentration of AEC from about 1 to about 2.5 mM. 43.The method of claim 1, wherein the transgenic soybean plant containsincreased levels of lysine compared to soybean plants which do notcomprise the DHPS encoding sequences.
 44. A method for generating aherbicide-resistant transgenic soybean plant comprising: introducinginto a soybean somatic embryogenic culture a polynucleotide encoding afunctional dihydrodipicolinate synthase (DHPS) polypeptide, operablylinked to an expression control sequence, wherein DHPS expressed fromthe introduced DHPS-encoding polynucleotide is effective to render anembryo resistant to selection-effective amounts ofS-2-aininoethylcysteine (2-AEC), and to render the plant resistant toherbicide-effective amounts of AEC, and contacting the embryo withselection effective amounts of 2-AEC.
 45. The method of claim 41,wherein the DHPS-encoding sequence encodes a bacterial DHPS that isresistant to AEC inhibition.
 46. The method of claim 41, wherein theDHPS-encoding sequence is isolated from soybean and has been geneticallyaltered to be resistant to AEC inhibition.
 47. The method of claim 41,wherein the DHPS encoding sequence comprises the sequence represented bySEQ. ID. NO:
 1. 48. A herbicide-resistant transgenic soybean plant, orprogeny thereof, comprising a polynucleotide encoding a functionaldihydrodipicolinate synthase (DHPS) polypeptide, operably linked to anexpression control sequence, wherein DHPS expressed from the introducedDHPS-encoding polynucleotide is effective to render the soybean plantresistant to herbicide-effective amounts of AEC, the plant having noantibiotic resistance marker.
 49. A soybean somatic embryo comprising anAEC resistant DHPS-encoding polynucleotide selected by the process ofclaim
 1. 50. A transgenic soybean plant which is free of apolynucleotide encoding a polypeptide imparting antibiotic resistanceselected by the process of claim
 1. 51. A transformation vectorcomprising a polynucleotide, operably linked to an expression controlsequence, encoding an AEC resistant DHPS and expressible in soybeansomatic embryos, a polynucleotide, operably linked to an expressioncontrol sequence, encoding a polypeptide of interest producing a desiredtrait upon expression in a soybean plant, and a polynucleotide impartinga trait selectable in bacterial culture, the vector having no antibioticresistance marker.
 52. The vector of claim 51, comprising a constitutivepromoter.
 53. The vector of claim 51, wherein the polynucleotideimparting a selectable trait is the DHPS-encoding sequence and thebacterial culture is an AEC-sensitive E. coli auxotroph.
 54. Atransgenic soybean plant comprising a polynucleotide encoding an AECresistant DHPS that is expressible in soybean somatic embryos and apolynucleotide encoding a protein imparting a desired trait in thesoybean plant or soybean, the plant being free of a polynucleotideencoding a polypeptide imparting antibiotic resistance.
 55. A soybeanseed produced by the soybean plant of claim
 51. 56. The plant of claim54, wherein the polypeptide imparting a desired trait is alysophosphatidate acyl transferase (LPAT) or diacylglycerolacyltransferase (DGAT).
 57. The plant of claim 54, wherein the plantproduces a soybean having increased oil content.
 58. The plant of claim54, wherein the polypeptide imparting a desired trait is a delta-9desaturase and/or a delta-12-desaturase.
 59. The plant of claim 54,wherein the delta-9 desaturase decreases saturated fatty acid contentsin the soybean plant and/or results in palmitoleic acid accumulation inthe soybean plant and/or high oleic acid soybean oil.
 60. The plant ofclaim 54, wherein the polypeptide of interest is one or more selectedfrom the group consisting of omega-3 desaturase, a polypeptide forimproved meal amino acid compositions, a disease resistance polypeptide,an insect resistance polypeptide, a nematode resistance polypeptide, aherbicide resistance polypeptide, a polypeptide affecting soybeancomposition or quality, a nutrient utilization polypeptide, anenvironmental or stress resistance polypeptide and a drought resistancepolypeptide, and combinations.
 61. The plant of claim 54, wherein theDHPS is a bacterial DHPS that is resistant to AEC inhibition.
 62. Theplant of claim 61, wherein the AEC resistant DHPS gene is obtained froma bacterium selected from Corynebacterium glutamicum and E. coli. 63.The plant of claim 54, wherein the DHPS gene is derived from soybean andis genetically altered to be resistant to AEC inhibition.
 64. The plantof claim 54, wherein the protein imparting the desired trait in theplant or bean is the DHPS.